Abstract:
Phosphoenolpyruvate carboxylase (PEPC) and malic enzyme
activities in soluble protein extracts of Avena coleoptiles
were investigated to determine whether their kinetics were
consistent with a role in cytosol pH regulation. Malic enzyme
activity was specific for NADP+ and Mn2+. Maximal labelled
product formation from [14C]-substrates required the presence
of all coenzymes, cofactors and substrates. Plots of rate
versus malate concentration, and linear transformations there-
2
of, indicated typical Michaelis-Menten kinetics at non-saturating
malate levels and substrate inhibition at higher malate levels.
pH increases between 6.5 and 7.25 increased near-optimal activity,
decreased the degree of substrate inhibition and the Kmapp(Mn2+)
but did not affect the Vmax or Kmapp(malate). Transformed data
of PEPC activity demonstrated non-linear plots indicative of
non-Michaelian kinetics. pH increases between 7.0 and 7.6 increased
the Vmax and decreased the Km app (Mg2+) but did not affect the
Kmapp(PEP). Various carboxylic acids and phosphorylated sugars
inhibited PEPC and malic enzyme activities, and these effects
decreased with pH increases. Metabolite inhibited malic enzyme
activity was non-competitive and resulted mainly from Mn2+
chelation. In contrast, metabolite inhibited PEPC activity was
unique for each compound tested, being variously dependent on the
PEP concentration and the pH employed.
These results indicate that fluctuations in pH and metabolite
levels affect PEPC and malic enzyme activities similarly and that
3
the in vitro properties of PEPC are consistent with its proposed
role in a pH-stat, whereas the in vitro properties of the malic
enzyme cannot be interpreted in terms of a role in pH regulation.
